Liquid Bridge and System

ABSTRACT

A bridge ( 30 ) comprises a first inlet port ( 31 ) at the end of a capillary, a narrower second inlet port ( 32 ) which is an end of a capillary, an outlet port ( 33 ) which is an end of a capillary, and a chamber ( 34 ) for silicone oil. The oil is density-matched with the reactor droplets such that a neutrally buoyant environment is created within the chamber ( 34 ). The oil within the chamber is continuously replenished by the oil separating the reactor droplets. This causes the droplets to assume a stable capillary-suspended spherical form upon entering the chamber ( 34 ). The spherical shape grows until large enough to span the gap between the ports, forming an axisymmetric liquid bridge. The introduction of a second droplet from the second inlet port ( 32 ) causes the formation of an unstable funicular bridge that quickly ruptures from the, finer, second inlet port ( 32 ), and the droplets combine at the liquid bridge ( 30 ). In another embodiment, a droplet ( 55 ) segments into smaller droplets which bridge the gap between the inlet and outlet ports.

INTRODUCTION

Field of the Invention

The invention relates to control of liquids at small volumes such as atthe microfluidic level.

Prior Art Discussion

Microfluidics is a technology which in simple terms refers to themicro-scale devices which handle small volumes of fluids—as small asmicro-, nano- and pico- and femtolitre volumes. Microfluidic deviceshave dimensions ranging from several millimetres to micrometers.Typically dimensions of the device are measured in micrometers. Giventhe small dimensions of microfluidic devices or components thereof, notsurprisingly microfluidic devices require construction and design whichdiffer from macro-scale devices. Simple scaling down in size ofconventional scale devices to microfluidic scale is not a simple option.Liquid flow in microfluidic devices differs from that of macro-scalesize devices. Liquid flow tends to be laminar, surface flux and surfacetension start to dominate and as a result effects not seen at the macrolevel become significant. At the microfluidic level other differencesinclude faster thermal diffusion, predominately laminar flow and,surface forces are responsible for capillary phenomena and an electricdouble layer (EDL).

Because microfluidics can accurately and reproducibly control anddispense minute volumes of fluid—in particular volumes less than one td.The application of microfluidics can have significant cost-savingpotential. The use of microfluidics technology can potentially reducecycle times, shorten time-to-results and increase throughput.Furthermore incorporation of microfluidics technology can, in theory,enhance system integration and automation.

Heretofore, handling of small quantities of liquids being transferredfrom one device or conduit to another often involves pipetting, which isa time-consuming manual procedure.

US2005/0092681 describes a device for mixing liquids by transportingthem to a confluent portion. US2005/0272144 discloses user of a mixingflow path for diffusion and mixing of liquids.

The invention is directed towards achieving improved liquid control inmicrofluidic systems.

SUMMARY OF THE INVENTION

In this specification the term “droplet” is used to mean a smallquantity or plug of liquid as it flows in an immiscible carrier liquidalong a conduit.

According to the invention, there is provided a liquid bridgecomprising:

-   -   at least one inlet port for delivery of a liquid A and an        immiscible carrier liquid B,    -   at least one outlet port,    -   a chamber within which the ports are located, the chamber having        a capacity such that it fills with carrier liquid B to fill the        space between an inlet port and an outlet port; and    -   wherein the ports are mutually located and have dimensions such        that liquid A periodically bridges to the outlet port due to        fluidic instability and droplets are periodically delivered to        the outlet port.

In one embodiment, the liquid bridge comprises at least two alignedinlet and outlet ports for droplet formation during flow of liquid fromone port to the other.

In one embodiment, the ports have a width dimension in the range of 150μm to 400 μm.

In one embodiment, the ports are circular in cross-section and saiddimension is diameter.

In one embodiment, at least one port is provided by an end of acapillary tube.

In one embodiment, the separation of an inlet port and a correspondingoutlet port is in the range of 0.2 mm and 2.0 mm.

In one embodiment, said separation is approximately 1.5 mm.

In another embodiment, the conduit and the ports allow passage of acarrier liquid B having a viscosity in the range of 0.08 Pas and 0.1Pas.

In one embodiment, the bridge comprises at least first and second inletports arranged for delivery of inlet liquid droplets and an output portfor outlet of a mixed liquid droplet.

In one embodiment, the first inlet port and the outlet port are co-axialand the second inlet port is substantially perpendicular to their axis.

In a further embodiment, the distance between the second inlet port andthe axis of the co-axial ports is in the range of 1.0 mm to 2.0 mm.

In one embodiment, the second inlet port has a smaller cross-sectionalarea than the first inlet port.

In one embodiment, an inlet port is arranged relative to an outlet portfor mixing of droplets of liquid A arriving at said inlet port bycollision within the chamber.

In another aspect, the invention provides a liquid bridge systemcomprising any liquid bridge as defined above, and a flow controller forcontrolling flow of liquids A and B to the bridge and operation of thebridge according to droplet formation characteristics.

In one embodiment, the controller directs flow of carrier liquid B at aflow rate in the range of 2 μl/min and 5 μl/min.

In one embodiment, the controller directs pressure in the chamber to bein the range of 0.5 bar and 1.0 bar above atmospheric.

In one embodiment, a droplet formation characteristic is a plot ofvolumetric ratio vs. droplet slenderness.

In a further embodiment, a droplet formation characteristic is capillaryseparation.

In one embodiment, a droplet formation characteristic is droplet volumevs. slenderness ratio.

In one embodiment, a droplet formation characteristic is V*(Q*) versusΛ*

In one embodiment, a droplet formation characteristic is ratio ofcapillary diameters.

In one embodiment, the carrier liquid B is oil.

In another embodiment, the controller comprises means for filling thechamber with carrier liquid B which is density matched with the liquid Asuch that a neutrally buoyant environment is created within the chamber.

In one embodiment, the carrier liquid B has characteristics causing thedroplets in the chamber to have a spherical shape between an inlet portand an outlet port.

In one embodiment, the density of the carrier liquid B and thecross-sectional areas of the ports are such that the carrier liquid Bflows and surrounds the droplets of liquid A in the outlet port.

In one embodiment, the velocity profile across the outlet port is suchas to cause internal movement within droplets of liquid as they flowfrom an outlet port.

In one embodiment, said internal flow is internal circulation.

In one embodiment, the carrier liquid B forms a protective film which isstatic very close to the internal surface of an outlet port and flowswith the droplets further from the said surface.

In one embodiment, a droplet formation characteristic is volumetricratio vs. funicular slenderness.

In one embodiment, the arrangements of the ports and the properties ofthe carrier liquid B are such as to cause segmentation of liquid A intoa plurality of outlet droplets.

In a further aspect, the invention provides a method of controllingoperation of any liquid bridge as defined above, the method comprisingdirecting flow of liquids A and B into the bridge with flowcharacteristics determined according to the geometry of the bridge portsand their separations and according to liquid properties, such thatliquid A periodically bridges to the outlet port due to fluidicinstability and droplets are periodically delivered to the outlet port.

In one embodiment, the flow is controlled to cause segmentation of acontinuous stream or large droplets of the liquid A in the chamber toprovide a sequence of segmented droplets derived from the inlet liquidA.

In one embodiment, different, miscible, liquids A and C are delivered tothe bridge, and flow is controlled so that droplets of liquids A and Cmix within the chamber.

In one embodiment, the liquids A and C are delivered to different inletports of the bridge, said ports being located so that droplets ofliquids A and C mix to form a single droplet which exits via the outletport.

In one embodiment, the liquids A and C are delivered to the bridge in asingle inlet port as successive droplets, and flow is controlled so thatthe droplets collide within the chamber to mix.

In one embodiment, the method comprises withdrawing carrier liquid Bfrom between said droplets before they enter the bridge so that they arecaused to collide within the chamber.

In one embodiment, the liquids A and C comprise different chemicalspecies contained within an aqueous phase.

In one embodiment, the carrier liquid B has a viscosity in the range of0.08 Pas and 0.1 Pas.

In one embodiment, the carrier liquid B flows into the bridge at a flowrate in the range of 2 μl/min and 5 μl/min.

In one embodiment, the carrier liquid B is density matched with theliquid A such that a neutrally buoyant environment is created within thechamber.

In one embodiment, the carrier liquid B has characteristics causing thedroplets in the chamber to have a spherical shape between an inlet portand an outlet port.

DETAILED DESCRIPTION OF THE INVENTION Brief Description of the Drawings

The invention will be more clearly understood from the followingdescription of some embodiments thereof, given by way of example onlywith reference to the accompanying drawings in which:—

FIGS. 1 and 2 are schematic diagrams of a PCR preparation microfluidicsystem of the invention;

FIGS. 3 and 4 are above and beneath perspective views of a pumpingsystem of the microfluidic system, and FIG. 5 is a cross-sectionaldiagram illustrating a sample well of the pumping system in more detail;

FIG. 6 is a sequence of diagrams A to G illustrating operation of amixing bridge incorporating a funicular bridge;

FIG. 7 is a diagram showing internal circulation and the protective oilfilm around a micro-reactor droplet;

FIG. 8 is a sequence of diagrams A to D illustrating operation of asegmentation bridge;

FIG. 9 is a sequence of photographs showing liquid dynamics anddimensions at a liquid bridge;

FIG. 10 is a diagram showing a characteristic plot of volumetric ratiovs. slenderness at a liquid bridge for segmentation;

FIG. 11 is a set of photographs of liquid bridge segmentors havingdifferent geometries;

FIG. 12 is a characteristic plot for a liquid bridge segmentor;

FIG. 13 is a collapsed data characteristic plot for liquid bridgesegmentation;

FIG. 14 is a set of photographs of three liquid bridge segmentors havingdifferent capillary radii;

FIGS. 15 and 16 are further characteristic plots for a liquid bridgesegmentor;

FIG. 17 is a photograph of a funicular bridge, also showing dimensionparameters;

FIG. 18 is a funicular liquid bridge characteristic stability plot; and

FIG. 19 is a set of diagrams A to D showing operation of an alternativemixer bridge of the invention.

DESCRIPTION OF THE EMBODIMENTS

Referring to FIGS. 1 and 2 a microfluidic preparation system 1 for aPolymerase Chain Reaction (“PCR”) system is shown at a high level. Itcomprises components 2-9 for, respectively flow control, sample loading,sample queuing, mixing, reaction segmentation, PCR thermocycling, RTfluorescence monitoring, and waste handling.

The system 1 is operated to find the gene expression profile of apatient sample using the PCR technique. Standard scientific protocolsare available on the extraction and purification of mRNA and thesubsequent production of cDNA. This is then mixed with the specificprimers and the general other reagents prior to amplification.

In the simple example illustrated sample streams of droplets A2, B2, C2,and D2 are managed in the queuing network 4 so that the order in whichthey are delivered to the mixer 5 is controlled as desired. For example,there may be a long sequence of droplets of type A2, followed byequivalent streams for B2, C2, and D2. Alternatively, there may berepeated sequences of A2, B2, C2, and D2, the droplets being interleavedin the queuing network 4. The mixer 5 joins together a droplet of sampletype D1 and each particular droplet which passes through the mixer 5.The chemical composition of the droplets is not the subject of thisinvention, suffice to say that they are chosen according to theparticular PCR experiment being run. The invention applies particularlyto the manner in which the droplets are mixed by the mixer 5 to providemicro reactor droplets. It also applies to the manner in which a longstream of a sample is broken up into a sequence of droplets of aparticular size by the segmentor 6. These droplets are separated by anenveloping carrier liquid, in this case oil A1 delivered to thesegmentor 6.

Thus, the system 1 achieves automation of the production of liquiddroplets in a carrier fluid with the sample, primers and reagents mixed.Take for example a situation where there are N genes, each interrogatedm times. To increase the experimental certainty, there may be m×Ndroplets. There will also be p housekeeping genes and q negativecontrols. The total number of droplets, M, may be calculated by:

M=(m×N)+p+q.

This may be of the order of 400 droplets.

Liquid bridges are used to mix the reagents and segment droplets in thecomponents 5 and 6. These generate a line of droplets carrying a varietyof different chemistries. The choice of fluid properties will bedescribed so that a liquid film is always present between the dropletand the channel wetted surfaces. This has the dual effect of preventingcarryover contamination and surface inhibitory effects that restrictamplification.

A typical Q-PCR reaction contains: fluorescent double-stranded bindingdye, Taq polymerase, deoxynucleotides of type A, C, G and T, magnesiumchloride, forward and reverse primers and patient cDNA, all suspendedwithin an aqueous buffer. Reactants, however, may be assigned into twobroad groups: universal and reaction specific. Universal reactants arethose common to every Q-PCR reaction, and include: fluorescentdouble-stranded binding dye, Taq polymerase, deoxynucleotides A, C, Gand T, and magnesium chloride. Reaction specific reactants include theforward and reverse primers and patient cDNA.

In more detail, the following describes the components 2-8.

-   2: A flow control system consisting of a precision pump with a motor    controlled drive that regulates the flow rates used to load and    drive samples through the system.-   3: Sample loading is accomplished by infusing oil into small wells.    These wells contain the universal reactants for each PCR reaction in    addition to specific primers. Each well contains a different primer    set to quantify specific gene expression levels. The samples exit    the wells as long microfluidic plugs.-   4: The loading process is followed by sample queuing where the    reactants flow through a network such that they are arranged    serially or alternatively as desired in a tube.-   5: The reactants then flow through the mixer where they are combined    with the final PCR reactant, the patient cDNA. The production of    negative reactants, containing no patient cDNA, is also possible    during this process.-   6: The relatively large reaction plugs are then segmented into    smaller plugs or droplets in a liquid bridge segmentation process.    This is performed to reduce experimental uncertainty.

Referring also to FIGS. 3 to 5, the wells labelled A1, A2, B1, B2, etc.in FIG. 2 connect directly to the flow control system 2, as shown inFIG. 3. The system 2 consists of eight Teflon-tipped plungers 10 eachinserted into a 686 μl cylinder in a polycarbonate infusion manifold 11filled with silicone oil. There is a substrate microchannel 21 formedbetween the manifold 11 and a cradle 12. The plungers 10, when driven ata constant velocity, pump the oil from the infusion manifold 11 throughthe sample wells and into the substrate microchannels. The flow ratesare maintained equal in each well so that the queuing of droplets onlydepends upon path length. The locations of the ports are shown in FIG.4. They connect directly to opposing substrate wells.

The plungers are driven by a pusher block (not shown) on a lead-screwconnected to a stepper motor to infuse silicone oil at equal flow ratesinto each sample well to pump the contents of the wells intomicrochannels. This method of pumping could also be done from areservoir, possibly with internal baffling to equalise the flow. In thiscase only a single plunger would be necessary.

The sample wells may be either integrated on disposable substrates orconnected via polycarbonate sample well strips on non-disposableversions. Alignment dowels 15 are used to position the wells accuratelyunder the infusion manifold 11. Sealing between mating components isensured with the use of elastomer gaskets 16 on each side of a samplewell strip 19. The gaskets provide a liquid-tight seal with theapplication of a constant upward force to the substrate cradle 12 and asealing sheet 20. The substrate measures 70 mm×90 mm in this embodiment.When an upward force is applied to the substrate cradle, a liquid tightseal is formed between the infusion manifold, sample well strip and themicrochannel substrate with the aid of elastomer gaskets at matinginterfaces.

As silicone oil is pumped by the plungers 10 it passes through the wells19 into which samples have been loaded. Thus, the flow into eachmicrochannel 21 is a stream of carrier oil and sample droplets for entryinto the queuing network 4.

Differences in microchannel path lengths delay the arrival of sampledroplets to the outlet of the queuing network 4. It can be seen fromFIG. 2, that the shortest path length exists from well A2 to the networkoutlet. The next shortest path length exists from well B2 to outlet andso on. The result of the queuing network is a linear array of reactiondroplets separated by silicone oil. This queuing network design has theadditional benefit of being scalable to array many samples.

The aqueous phase reaction droplets are combined with patient cDNA atthe mixer 5. The two-phase nature of microfluidic flow necessitates ameans of combining fluids. The fluids are combined into a single dropletin the mixer 5 by virtue of funicular bridge instability. FIG. 6 showsfunicular bridge mixing in a particular funicular bridge 30 of the mixer5. The bridge 30 comprises a first inlet port 31 at the end of acapillary, a narrower second inlet port 32 which is an end of acapillary, an outlet port 33 which is an end of a capillary, and achamber 34 for silicone oil. Initially, the entire system is primed witha density matched oil. The inlet and outlet ports 31 and 33 are ofdiameter 200 μm, more generally preferably in the range of 150 μm to 400μm. The separation of the ports 31 and 33 is c. 1 mm, and the distancebetween the second inlet port 32 and the axis of the ports 21 and 33 isc. 1.5 mm. The chamber 34 is 5 mm in diameter and 3 mm in depth. Theenveloping oil provides a pressure of no more than 0.5 to 1.0 bar aboveatmospheric. It has a viscosity of 0.08 to 0.1 Pas, and the flow rate isin the range of 2 to 5 μl/mn

The oil is density-matched with the reactor droplets such that aneutrally buoyant environment is created within the chamber 34. The oilwithin the chamber is continuously replenished by the oil separating thereactor droplets. This causes the droplets to assume a stablecapillary-suspended spherical form upon entering the chamber 34, steps Band C. The spherical shape grows until large enough to span the gapbetween the ports, forming an axisymmetric liquid bridge.

The introduction of a second droplet from the second inlet port 32causes the formation of an unstable funicular bridge that quicklyruptures from the, finer, second inlet port 32, causing all the fluid tocombine at the liquid bridge 30.

The sequence of illustrations A to G in FIG. 6 show how the envelopingliquid controls droplet formation and mixing according to surfacetension. The pressure in the chamber 34 is atmospheric. The interfacialtension within the chamber 34 is important for effective mixing. Also,the relative viscosity between the aqueous and oil phases is important.The internal pressure (Laplace pressure) within each droplet isinversely proportional to the droplet radius. Thus there is a higherinternal pressure within the droplet at the second inlet port 32.Because they are of the same phase, there is little interfacial tensionbetween the large and small droplets, and the internal pressures cause ajoining of the droplets, akin to injection of one into the other. Also,physical control of the locations of the droplets is achieved by theenveloping oil, which is of course immiscible with the droplets. Furtheraddition of a surfactant to either phase can change the interfacialtension.

FIG. 7 shows the internal circulation that takes place within a flowingdroplet 40 carried by an immiscible oil 41. The velocity profile on theright shows the velocity distribution within the plug relative to theaverage velocity of the flow. This internal circulation causes excellentmixing and enhances chemical reactions within the droplet 40, and henceit may be regarded as a micro-reactor. The location of a protective oilfilm 41 is also shown, separating the droplet 40 from the channel walls42 in addition to separating the droplets 40 from each other. Thepatterns observed may be visualised if the observer adopts a referenceframe that moves at the mean velocity of the flow. With this in mind,the flow can be imagined as a fast moving fluid along thecentre-streamline toward the leading face of the plug. Fluid is thencirculated back to the rear of the plug near the walls of themicrochannel. Internal circulation within flowing microfluidic plugs isa powerful mixing mechanism that, in contrast to existing threedimensional serpentine micromixers, does not require complexmicrochannel geometries. Homogenous mixing is known to increase reactionkinetics and internal circulation is an important advantage of thetwo-phase plug flow regime. The establishment of internal circulationand protective films is enhanced by the use of circular polymericmicrochannels, specifically FEP fluorocarbon polymeric microchannels.This mechanism enhances mixing within a droplet downstream of the mixer5.

Fully mixed plugs then enter the segmentor 6 to split master reactionplugs into four smaller droplets, containing identical chemistries thatare individually monitored during thermocycling. This step reducesexperimental uncertainty. A bridge 50 of the segmentor 6 is shown inFIG. 8. The bridge 50 comprises an aqueous, inlet 51, an oil inlet 52,an outlet 53, and a chamber 54. The chamber 54 is 5 mm in diameter and 3mm in depth and the internal pressure caused by flow of silicone oil isno more than 0.5 to 1.0 bar above atmospheric. The diameter of the ports51 and 53 is 200 μm, and is more generally preferably in the range of150 μm to 400 μm. The spacing between the ports 51 and 53 is 0.5 mm, andis more generally preferably in the range of 0.2 to 1.0 mm. The outletflow rate from the segmentor is 5 μl/min, more generally in the range of2 to 8 μl/min.

The liquid bridge's geometry and the enveloping carrier liquid create aperiodic instability between the opposing ports 51 and 52 due to surfacetension. A droplet 55 is initially formed at the end of the inlet port51 (diagram A). As shown in diagram B the droplet liquid thenmomentarily bridges the ports 51 and 53. The volume held in this bridgeis then steadily reduced by the action of the silicone oil inlet. Thiscauses the formation of an unstable liquid bridge that ruptures torelease a smaller plug at the outlet. When the inlet oil flowratematches the aqueous droplet flowrate, smaller segmented droplets,separated by the same volume of silicone oil, are produced by thisbridge. The segmenting mechanism reliably produces uniform aqueous plugsseparated by oil that do not rely on the shear force exerted by thecarrier fluid.

Multiple dispensing bridges with N inputs 51 and N outputs 53 are alsoprovided. In this case multi lumen tubing may be used as a conduit tocarry fluid from the preparation system through the continuous flow PCRthermocycler. Multi lumen tubing contains many micro-bores such thateach bore represents a fluid path through which PCR thermocycling mayoccur.

Liquid bridge stability was studied as a means to predicting thegeometric conditions at which rupture occurs. Liquid bridge rupture maybe defined as the complete breakage of the liquid filament connectingone solid support to the other. The dimensionless parameterscharacterising liquid bridges are used to define the stability boundaryat which rupture was observed. FIG. 9 presents images of liquid bridgesat three slenderness conditions just prior to rupture. The rupture wascaused by the withdrawal of liquid bridge fluid from one capillary tube.It was observed that low slenderness ratio liquid bridges, an example ofwhich is shown in FIG. 9 (A), adopt a thimble shape at the minimumvolume stability. Larger slenderness ratio liquid bridges, such as thatshown in FIG. 9 (C), possess a barrel form with a maximum radius at thebridge mid-span. Intermediate slenderness ratios were found to have anear cylindrical shape at the minimum volume stability limit. Images(A), (B) and (C) of FIG. 9 show liquid bridges with slenderness ratiosof 1.09, 2.45 and 6.16 respectively.

The stability of liquid bridges was examined as a function ofslenderness, Λ*, which is the ratio of tip separation, L, to the meandiameter, 2R₀, of the supporting capillaries, i.e. Λ*=L/2R₀. Stabilitywas also investigated as a function of volumetric ratio, V*, which isthe ratio of liquid bridge volume to the volume of a cylinder with aradius R₀, the average radius of the supporting capillaries, i.e.V*=V/(πR₀ ²L). The location of the stability boundary, or rupture point,was determined experimentally by fixing the slenderness, establishing astable liquid bridge between capillary tips and withdrawing fluid fromone capillary until rupture was observed. A digital image of the liquidbridge just prior to rupture was then analysed, using an edge detectionmeasurement technique to determine the total volume and hence thevolumetric ratio, V*. The slenderness was then adjusted and theexperiment repeated. K* represents the ratio of the radius of thesmaller disk, R₁, to the radius of the larger one, R₂, that is K*=R₁/R₂.FIG. 12 shows the approximate location of the minimum volume stabilityboundary for liquid bridges with a lateral Bond number of 1.25×10⁻⁴, anear weightless environment. Vertical and horizontal error bars indicateexperimental uncertainty.

At high volumetric ratios, FIG. 9 (C) for example, bridges maintaintheir integrity and reach a minimum energy configuration. At lowvolumetric ratios, FIG. 9 (A) for example, the bridges break before theinterfacial energy is minimized. The initial dip in the stabilityboundary at low slenderness ratios is caused by low-volume droplets notfully wetting the exposed fused silica of the capillary tips. Theinfluence of unequal capillaries on the Λ*−V* stability diagram is alsoshown in FIG. 10. It can be seen that the unstable region of the Λ*−V*plane increases as the parameter K*, the ratio of capillary radii,decreases. The results presented in FIG. 10 confirmed that the staticstability of liquid bridge is purely geometrical at low Bond numbers. Itis notable that low slenderness ratio bridges are almost completelystable, with respect to rupture, for all capillary radii measured.Rupture was observed only at very low volumetric ratios with the liquidbridge assuming a thimble shape. Liquid bridge instability when appliedto fluid dispensing is particularly useful as a replacement formicrochannel shear-based dispensing systems. In more detail, FIG. 10shows an experimentally determined stability diagram for a de-ionizedwater liquid bridge in a density matched silicone oil, Bond number:1.25×10⁻⁴. Vertical error bars indicate the volumetric ratio uncertaintyas a result of camera frame rate. Horizontal error bars indicateslenderness uncertainty due to capillary tip misalignment. The parameterK* is the ratio of supporting capillary radii.

The following describes the use of liquid bridge instability as amechanism for dispensing sub-microlitre volumes of fluid in a continuousmanner. The approach uses the liquid bridge's dependence on geometry tocreate a periodic instability between opposing capillary tips. Thedispensing mechanism provides a reliable means of producing uniformaqueous plugs separated by silicone oil that did not rely on the shearforce exerted by the carrier fluid. The repeatability with which themethod can dispense plugs is examined. The approach uses the liquidbridge's dependence on geometry to create a periodic instability betweenopposing capillary tips. A stable liquid bridge is first establishedbetween aqueous inlet and outlet. The volume held in this bridge is thensteadily reduced by the action of the silicone oil inlet. This causesthe formation of an unstable liquid bridge that ruptures to release asmaller plug at the outlet. The segmenting mechanism provides a reliablemeans of producing uniform aqueous plugs separated by silicone oil thatdoes not rely on the shear force exerted by the carrier fluid.Furthermore, a protective oil film is established between the walls ofthe circular capillaries and the droplet to prevent carryovercontamination.

FIG. 11 (A)-(D) presents images of liquid bridge dispensing at fourdifferent slenderness ratios. (A) Λ*=0, (B) Λ*=0.76, (C) Λ*=1.37 and (D)Λ*=2.31. Q*=0.5, K*=0.44. Increasing the capillary tip separation, andhence the slenderness ratio increases the plug volumes dispensed. Q*,the oil flowrate as a fraction of the total flowrate, was maintainedconstant at 0.5. Image (A) shows dispensing with the dispensingcapillary inserted inside the outlet capillary. This configuration wasassigned a slenderness ratio, Λ*, of zero. Slenderness ratios close tozero resulted in the smallest volume plugs dispensed for this geometry.The effect of increasing tip separation on dispensed plug volume isshown in FIG. 11 (B)-(D). Increasing tip separation, i.e. slendernessratio, resulted in larger volume aqueous plugs punctuated byapproximately the same volume of silicone oil. This was due to thesilicone oil inlet flowrate being maintained constant and equal to theaqueous droplet inlet flowrate.

FIG. 12 presents a plot of V*, against slenderness ratio, Λ*, where V*is the dimensionless plug volume scaled with R₀ ³, i.e. V*=V/R₀ ³.Results are presented for three different values of the oil flowratefraction, Q*, with the ratio of capillary tip radii, K*, maintainedconstant at 0.44. The axis on the right-hand side of the plot indicatesthe measured plug volume. Horizontal error bars indicate slendernessuncertainty as a result of positional inaccuracy. Vertical error bar area result of uncertainty in the plug volume calculation due to imageprocessing. The results show the expected trend of increased plug volumewith liquid bridge slenderness ratio. Decreasing Q* resulted in adramatic increase in dimensionless plug volume. Altering Q* alsoaffected the volume of silicone oil separating the aqueous plugs as Q*is the oil flowrate as a fraction of the total flowrate. The lowestrepeatable volume measured using this particular geometry wasapproximately 90 nL with Λ*=0, Q*=0.75. The highest volume measured wasapproximately 3.9 μL with Λ*=2.36, Q*=0.25.

In flows where the non-wetting fluid, i.e. the aqueous phase, isdisplaced by wetting fluid, i.e. oil, a thin film of the wetting fluidseparates the droplets from the capillary surface. The thickness of thefilm results from a balance between the oil viscosity, η, and theinterfacial tension, σ_(i). The thickness of the oil film deposited in acapillary of radius r is given by;

h=1.34r(Ca ^(2/3))  (0.1)

The capillary number, Ca, is given by:

$\begin{matrix}{{{Ca} = \frac{\eta \; U}{\sigma_{i}}},} & (0.2)\end{matrix}$

where U represents the mean velocity of the flow. Equation (0.1) isobeyed if the film is thin enough to neglect geometric forces, h<0.1r,and thick enough to avoid the influence of long range molecularattraction, h>100 nm. Typical oil film thicknesses for plug flow through400 μm polymeric fluorocarbon internal diameter tubing were calculatedto be of the order of 1 μm. This film thickness was too small to resolvewith any degree of accuracy from experimental images. The oil film does,however, form a protective coating preventing aqueous reactor fluid fromcontacting the Teflon tubing. This has the advantage of preventing amechanism responsible for carryover contamination whereby small dropletsmay be deposited onto the walls of microchannels. Table 1 below presentstwo examples of oil-surfactant combinations used to successfullyestablish protective oil films around flowing droplets. Surfactantadditives act to change the interfacial tension between droplets and theoil carrier fluid such as to promote the establishment of a protectiveoil film, the thickness of which is given by Equation 0.1.

TABLE 1 Oil Surfactant Concentration FC40 1H,1H,2H,2H-Perfluoro-1- 2%W/V decanol AS100 Silicone Oil Triton X-100 0.1% V/V in PCR BufferSolution

FIG. 13 presents a dimensionless plot of the product of V* and Q* versusΛ*. The data, taken from the plot shown in FIG. 12, collapsed on to thetrend line within the bounds of uncertainty. The data applies togeometries with K*=0.44.

Notwithstanding this geometric constraint, the collapsed data does yieldvaluable design information. Consider a microfluidic system designerdeciding on an appropriate geometry for a segmenting device. Thedesigner will usually know the exact volume to dispense from the outlinespecification for the device. If there is a sample frequencyrequirement, the designer may also know a value for Q*. Recalling thatK*=R₁/R₂, where R₁ and R₂ are the inlet and outlet diametersrespectively makes the design process relatively easy. Deciding on anarbitrary value for an outlet diameter fixes the aqueous inlet diameteras the data shown in FIG. 15 applies to only to geometries with K*=0.44.With this information in hand, an appropriate value for V*(Q*) may becalculated. The corresponding value for Λ* may then be read from thedesign curve shown in FIG. 13. Finally, Λ* may be used to calculate thetip separation between the inlet and outlet.

As mentioned previously, the data presented in FIGS. 12 and 13 appliesto geometries with K*=0.44. The effect of altering K* on plug volumesdispensed was also investigated. Images of liquid bridge dispensing atthree different values for K* are presented in FIG. 14. Images (A), (B)and (C) correspond to K* values of 0.25, 0.44 and 1.0 respectively. A K*value of 0.25 was achieved by assembling a 200 μm fused silicamicrocapillary at the end of a polymeric capillary tube by a reductionof internal diameter through appropriately sized fused silica. Sealingwas ensured with the addition of cyanoacrylate glue at the sleeveinterfaces.

FIG. 15 presents a dimensionless plot of V* versus Λ* for threedifferent values of K*. The dimensionless plug volume, V*, was scaledwith R₂ ³, and not R₀ ³ as previously. This permitted a directcomparison of dimensionless plug volumes as R₂ remained constantthroughout the experiment. It can be seen that decreasing K* generallylowers the plug volumes dispensed for any given value of slenderness,Λ*. The minimum volume dispensed with K*=0.25 was approximately 60 nLwhilst that of K*=0.44 and K*=1 was approximately 110 nL. Attempts tocollapse the data shown in FIG. 15 onto a single line, similar to theplot shown in FIG. 13, were unsuccessful. This was due to the highlynon-linear relationship between K* and V* for any given value of Λ*.

The repeatability with which the liquid bridge dispensing system coulddeliver fluid was of particular interest. FIG. 16 plots plug volumevariation over fourteen measurements for a dispensing system withK*=0.44. The results show mean plug volumes of approximately 120 nL and56 nL with maximum volumetric variations of ±4.46% and ±3.53%respectively. These volumetric variations compare favourably tocommercial available micropipettes which have an uncertainty of ±12%when dispensing 200 nL. The accuracy with which one may dispense usingmicropipettes, however, is thought to be largely dependant upon userskill. The automation of dispensing systems may therefore be justifiedas a means of eliminating user-user variability. The volumetric analysispresented in FIG. 16 shows liquid bridge dispensing to be a veryrepeatable means of continuously dispensing sub-microlitre volumes offluid.

Referring again to the funicular bridge of FIG. 6 an example ispresented in FIG. 17. The bridge consists of two opposing capillaries ofthe same external diameter. The second inlet part is of a finercapillary orientated at right angles to and situated half-way betweenthe other two capillaries. Constraints on opposing capillary radius andthe placement of the third capillary helped to simplify thedimensionless stability study. The investigation also necessitatedmodifications to the dimensionless parameters characterisingaxisymmetric liquid bridge geometry. The slenderness ratio, Λ*, wascalculated using:

$\begin{matrix}{{\Lambda^{*} = \frac{\sqrt{L^{2} + S^{2}}}{2R_{0}}},} & (1)\end{matrix}$

where L and S correspond to the distances indicated in FIG. 17. R₀ isdefined as the mean radius, i.e. (R₁+R₂)/2. K* is defined as R₁/R₂. Thevolumetric ratio, V*, is defined as:

$\begin{matrix}{{V^{*} = \frac{\overset{\_}{V}}{\pi \; R_{0}^{2}\sqrt{L^{2} + S^{2}}}},} & (2)\end{matrix}$

where V is the measured volume at which bridge collapse occurs. In termsof the geometry presented in FIG. 17, a funicular bridge collapsecorresponds to detachment from the finer capillary.

FIG. 18 shows a stability diagram the approximate location of theminimum volume stability boundary for purified water funicular liquidbridges with a lateral Bond number of 1.25×10⁻⁴, a near weightlessenvironment. The boundaries of stability were found by fixing a valuefor Λ*, establishing a stable funicular bridge and withdrawing fluiduntil the bridge collapsed. The collapse was recorded via a CCD and theframe immediately following rupture was analysed to measure the volume.The calculation of the bridge volume was simplified by the fact that thecollapsed funicular bridge exhibited axisymmetry with respect to theaxis of the two larger capillaries. Minimum volume stability boundariesare plotted for K*=0.25 and K*=0.44. Lower K* values displayed increasedinstability. Volumetric data for Λ* values lower than approximately 1.5was difficult to obtain with the geometry used and so was omitted fromthe stability diagram. This is, to the best of the inventor's knowledge,the first experimental study of funicular bridges for this application.The formation of a funicular bridge deemed unstable by the graph shownin FIG. 18 ensures the injection of fluid into an aqueous plug passingthrough opposing capillaries. A further advantage to using funicularbridge dispensers is based on the speed at which the process takesplace. Typical instabilities last of the order of 100 ms, insufficienttime for the host droplet fluid to diffuse to the dispensing capillarytip. This is a further preventative measure against carryovercontamination.

The two input one output, funicular bridge can be configured so that theexpression profile of many genes may be addressed. One input containsthe primer and premix in a continuous phase, the outlet then deliversthem in droplet form. Firstly many input and output capillaries, say p,can be set in planes perpendicular to that of FIG. 6. A perpendiculararrangement allows for good optical access in the planar thermal cyclerwhich is connected to the output. Each arrangement of two inputs and oneoutput can be used to address a single primer, giving p primers. This,however, would make for a very long device in the plane perpendicular toFIG. 6. If serially variant primers were fed into each input, asdescribed in FIGS. 2 and 8, numbering q, this would reduce the scale.Further, if the primers were multiplexed, to order r, in each dropletthe scale would be further reduced. The number of primers that couldthen be addressed would be:

N=p×q×r.

By this means a PCR test of the whole genome of any living form,including the human, could be addressed, which would have applicationsbeyond diagnosis, in many fields of pure and applied science.

In another embodiment, mixing of droplets may be achieved with only oneinlet port and two outlet ports. Inlet droplets are close together, andthe delay for droplet formation within the chamber due to a reduction influid flow through the main line causes a collision and hence mixing.Such mixing may be caused by withdrawal of oil from the chamber, orupstream of it. Referring to FIG. 19 a bridge 60 has an inlet port 61,an outlet port 62, an oil withdrawal port 63, and a chamber 64. A largeleading droplet entering the chamber forms a droplet 65 in the chamber.As oil is withdrawn from the chamber 64 through the oil withdrawal port63, a smaller trailing droplet collides with the leading droplet bubble65 so that the mixing occurs. A larger mixed droplet 67 leaves via theoutlet port 62.

In more detail, initially, the entire system is primed with a densitymatched oil. The diameter of the ports 61, 62 and 63 is 200 μm, and ismore generally preferably in the range of 150 μm to 400 μm. The spacingbetween the ports 61 and 62 is c. 1 mm, and is more generally preferablyin the range of 0.2 to 1.5 mm. The enveloping oil is controlled to havea pressure of no more than 0.5 to 1.0 bar above atmospheric. Theenveloping liquid is silicone oil with a viscosity of 0.08 to 0.1 Pas.

As with the lateral mixing bridge 30 and the segmentation bridge 50,droplets are enveloped by carrier oil entering and exiting the bridge 60via a protective oil film around the droplets. This provides anon-contacting solid surface that prevents carryover contamination fromone droplet to the other. The oil is used as the control fluid and isdensity-matched with the reactor plugs such that a neutrally buoyantenvironment is created within the oil chamber 64. When two unmixeddroplets arrive at the chamber in series from the inlet port 61, thefirst droplet assumes a stable capillary-suspended spherical form uponentering the chamber, step A. The spherical shape grows until largeenough to span the gap between the ports, forming an axisymmetric liquidbridge, step B. The control outlet port 63 removes a flow of oil fromthe chamber causing the first droplet to slow and remain as a sphericalshape at the outlet port 62 for a longer period. This allows time forthe second droplet to form a stable capillary-suspended spherical shapeon entering the chamber. With the first droplet formed as a sphericalshape at the outlet 62 and the second droplet formed as a sphericalshape at the inlet, the droplets can form as one and create anaxisymmetric liquid bridge, step C. The mixed droplet then exits throughthe outlet port 62, step D.

The flow conditions most be matched so that the flow through the inlet61 is greater than the flow out of the control outlet port 63. A typicalflow in through the inlet is typically 5 μl/mn and more generally in therange of 2 to 7 μl/mn. The flow away from the chamber through thecontrol port 63 is typically 2.5 μl/mn and more generally in the rangeof 1 to 5 μl/mn. Since there is conservation of mass flow within thebridge, this means that the flow through the outlet port 62 will balancethe bridge to give a flow of typically 2.5 μl/mn and more generally inthe range of 1 to 5 μl/mn.

This serial mixing bridge 60 can be used with a constant outlet flowthrough the control port 63. In doing so not only will droplets be mixedbut also the fluid flow through the system can be decreased. Inaddition, this bridge 60 can be used in conjunction with a sensor totime the withdrawal of fluid through the control port 63 so as tomaintain the main fluid flow at a generally constant flowrate. Thesensor used can be a droplet detection sensor which comprises of a LEDand photodiode. The LED is projected directly onto the centre of thetube. A photodiode is positioned directly opposite the LED to pick upthe light refracted through the tube. As a droplet of varying propertiesto that of oil flows past the LED and photodiode, the light refractedthrough the liquid is altered slightly. This slight alteration isdetected by the photodiode in the form of a change in voltage. Thischange in voltage can be used to time the control flow through outletport 63. The serial mixing bridge 60 can also be used downstream of thelateral mixing bridge 30. In doing so, the system can compensate fordroplets that have not yet mixed after flowing through the lateral mixer30. The reason for droplets not mixing may be that the droplets are outof phase with each other and have not met simultaneously at the lateralmixing bridge 30. Another system of ensuring mixing has occurred is toinclude a droplet detection sensor with the serial mixing bridge 60downstream of the lateral mixing bridge 30. If the droplet detectionsenses two droplets in unusually quick succession then the droplets havenot yet mixed. The droplet detection sensor can then switch on theserial mixer 60 and mix the two droplets.

It will be appreciated that the invention provides for particularlyeffective processing of droplets for applications such as amplificationof nucleic acids. It provides particularly effective mechanisms formixing of droplets and for segmenting long droplets or plugs.

The invention is not limited to the embodiments described but may bevaried in construction and detail. For example, the sample liquid may bedelivered continuously to an inlet port in some embodiments, forsegmentation. In this case the same basic mechanism provides droplets ofa controlled size in to the outlet port. Also, the flow controller maymerely be a passive feed system based on gravity or indeed capillaryaction rather than an active pumping means such as an infusion pump.

1. A liquid bridge comprising: at least one inlet port for delivery of aliquid A and an immiscible carrier liquid B, at least one outlet port, achamber within which the ports are located, the chamber having acapacity such that it fills with carrier liquid B to fill the spacebetween an inlet port and an outlet port; and wherein the ports aremutually located and have dimensions such that liquid A periodicallybridges to the outlet port due to fluidic instability and droplets areperiodically delivered to the outlet port.
 2. The liquid bridge asclaimed in claim 1, wherein the liquid bridge comprises at least twoaligned inlet and outlet ports for droplet formation during flow ofliquid from one port to the other.
 3. The liquid bridge as claimed inclaim 1, wherein the ports have a width dimension in the range of 150 μmto 400 μm, the ports are circular in cross-section and said dimension isdiameter, and at least one port is provided by an end of a capillarytube.
 4. (canceled)
 5. (canceled)
 6. The liquid bridge as claimed inclaim 1, wherein the separation of an inlet port and a correspondingoutlet port is in the range of 0.2 mm and 2.0 mm or is approximately 1.5mm.
 7. (canceled)
 8. The liquid bridge as claimed in claim 1, wherein aconduit and the ports allow passage of a carrier liquid B having aviscosity in the range of 0.08 Pas and 0.1 Pas.
 9. The liquid bridge asclaimed in claim 1, wherein the bridge comprises at least first andsecond inlet ports arranged for delivery of inlet liquid droplets and anoutput port for outlet of a mixed liquid droplet.
 10. The liquid bridgeas claimed in claim 9, wherein the first inlet port and the outlet portare co-axial and the second inlet port is substantially perpendicular totheir axis, the distance between the second inlet port and the axis ofthe co-axial ports is in the range of 1.0 mm to 2.0 mm, and the secondinlet port has a smaller cross-sectional area than the first inlet port.11. (canceled)
 12. (canceled)
 13. (canceled)
 14. A liquid bridge systemcomprising: A liquid bridge that comprises: at least one inlet port fordelivery of a liquid A and an immiscible carrier liquid B, at least oneoutlet port, a chamber within which the ports are located, the chamberhaving a capacity such that it fills with carrier liquid B to fill thespace between an inlet port and an outlet port; and wherein the portsare mutually located and have dimensions such that liquid A periodicallybridges to the outlet port due to fluidic instability and droplets areperiodically delivered to the outlet port; and a flow controller forcontrolling flow of liquids A and B to the bridge and operation of thebridge according to droplet formation characteristics.
 15. The system asclaimed in claim 14, wherein the controller directs one or more of aflow of carrier liquid B at a flow rate in the range of 2 μL/min and 5μL/min and a pressure in the chamber to be in the range of 0.5 bar and1.0 bar above atmospheric.
 16. (canceled)
 17. The system as claimed inclaim 14, wherein a droplet formation characteristic is one or more of aplot of volumetric ratio vs. droplet slenderness, a capillaryseparation, a droplet volume vs. slenderness ratio, V*(Q*) versus Λ*, aratio of capillary diameters, and volumetric ratio vs. funicularslenderness.
 18. (canceled)
 19. (canceled)
 20. (canceled)
 21. (canceled)22. (canceled)
 23. The system as claimed in claim 14, wherein thecontroller comprises means for filling the chamber with carrier liquid Bwhich is density matched with the liquid A such that a neutrally buoyantenvironment is created within the chamber.
 24. The system as claimed inclaim 14, wherein the carrier liquid B has characteristics causing thedroplets in the chamber to have a spherical shape between an inlet portand an outlet port.
 25. The system as claimed in claim 14, wherein thedensity of the carrier liquid B and the cross-sectional areas of theports are such that the carrier liquid B flows and surrounds thedroplets of liquid A in the outlet port.
 26. The system as claimed inclaim 14, wherein the velocity profile across the outlet port is such asto cause internal movement within droplets of liquid as they flow froman outlet port.
 27. (canceled)
 28. (canceled)
 29. (canceled)
 30. Thesystem as claimed in claim 14, wherein the arrangements of the ports andthe properties of the carrier liquid B are such as to cause segmentationof liquid A into a plurality of outlet droplets.
 31. A method ofcontrolling operation of a liquid bridge as claimed in claim 1, themethod comprising directing flow of liquids A and B into the bridge withflow characteristics determined according to the geometry of the bridgeports and their separations and according to liquid properties, suchthat liquid A periodically bridges to the outlet port due to fluidicinstability and droplets are periodically delivered to the outlet port.32. The method as claimed in claim 31, wherein the flow is controlled tocause segmentation of a continuous stream or large droplets of theliquid A in the chamber to provide a sequence of segmented dropletsderived from the inlet liquid A.
 33. The method as claimed in claim 31,wherein different, miscible, liquids A and C are delivered to thebridge, and flow is controlled so that droplets of liquids A and C mixwithin the chamber and liquids A and C comprise different chemicalspecies contained within an aqueous phase.
 34. The method as claimed inclaim 33, wherein the liquids A and Care delivered to different inletports of the bridge, said ports being located so that droplets ofliquids A and C mix to form a single droplet which exits via the outletport.
 35. The method as claimed in claim 33, wherein the liquids A and Care delivered to the bridge in a single inlet port as successivedroplets, and flow is controlled so that the droplets collide within thechamber to mix such that carrier liquid B is withdrawn from between saiddroplets before they enter the bridge so that they are caused to collidewithin the chamber.
 36. (canceled)
 37. (canceled)
 38. (canceled) 39.(canceled)
 40. (canceled)
 41. (canceled)